The present invention generally relates to liquid chromatographs and more particularly to a liquid chromatograph that uses a semi-micro column or micro column.
Liquid chromatographs are used extensively as a means for separating and analyzing chemical substances. Particularly, a liquid chromatograph that uses semi-micro column or micro column having an inner diameter of 1-2 mm or less is studied intensively in view of advantageous feature of high sensitivity, high resolution and high precision analysis.
In the art of liquid chromatography, the so-called gradient elution is employed commonly, wherein the composition of the solvent flowing through the column is changed with time. In the gradient elution, it is particularly necessary to control the composition of the solvent exactly, and because of this, a mixer is used for mixing a plurality of solvents.
When using semi-micro-columns having a small diameter for the gradient elution, it is necessary to set the flow rate of the fluid acting as the mobile phase in the column to be small, in the order of one-fifth to one-tenth or less, as compared with the flow rate employed in the conventional liquid chromatographs. Associated with this, there occur various problems to be solved.
FIG. 1 is a block diagram showing the schematic construction of a conventional liquid chromatograph designed for the gradient elution.
Referring to FIG. 1, the liquid chromatograph includes a first pump 11A for pumping a first solvent A and a second pump 11B for pumping a second solvent B, wherein the pumps 11A and 11B, respectively, supply the solvents A and B to a mixer 13 under control of a system controller 12. After mixing in the mixer 13 to a desired mixing ratio, the mixture of the solvents A and B are supplied to a sampler 14 wherein a sample solution held in a syringe 15 is injected to the mixture thus formed. The sample solution contains various chemical substances to be analyzed. Thereafter, the sample solution is supplied, together with the solvent A and/or B, to a column 16. Upon passage through the column 16, chemical substances in the sample solution are separated and supplied to a detector 17 together with the solvent. The detector 17, in turn, carries out a qualitative and/or quantitative analysis of the chemical species contained in the solvents A and/or B. After analysis in the analyzer 17, the solvents and the chemical substances are ejected to a waste reservoir 18.
FIGS. 2A and 2B show various constructions of the mixer 13 used in the liquid chromatograph of FIG. 1.
Referring to FIG. 2A, the mixer 13 mixes the solvents A and B in a chamber 13b by means of a rotating stirrer 13a. However, the mixer 13 of FIG. 2A tends to create a dead space 13x along the vessel wall of the chamber 13b wherein no substantial mixing occurs. In the low flow rate system such as the semi-micro-column liquid chromatograph, the effect of the dead space 13x appears particularly conspicuous.
FIG. 2C shows the mixing characteristic of the mixer 13 of FIG. 2A for a case in which methanol is used for the solvent A and a mixture of methanol and a small amount of acetone (0.05 volumetric percent) is used for the solvent B. In the measurement of the mixing characteristic, the mixing ratio is controlled stepwise according to a program curve shown at the left of FIG. 2C while simultaneously detecting the acetone content in the solvents thus mixed. The curve at the right of the program curve represents the actual or measured mixing ratio.
In the experiment, only the solvents A and B are caused to flow through the column 16 under the total flow rate set to 200 .mu.m/min. Further, the detection of acetone is made by measuring the ultraviolet absorption at the wavelength of 245 nm. In the example of FIGS. 2A and 2C, it will be noted that there occurs a momentary drop in the measured mixing ratio when the programmed mixing ratio is increased linearly and subsequently held at a level of 50%. Further, it should be noted that the actual characteristic curve at the left of the program curve generally has a rounded shoulder, indicating that the mixing cannot follow the program control.
The mixer 13 of FIG. 2B is proposed for eliminating the foregoing problem of the mixer of FIG. 2A.
Referring to FIG. 2B, the chamber 13b of the mixer is filled with small beads 13c, and the solvents A and B are caused to flow through irregular paths of fluid formed between the beads. By employing the construction as such, the problem of irregular mixing ratio associated with the stepwise change of the program mixing ratio is successfully eliminated as indicated in FIG. 2D, wherein FIG. 2D shows the result of measurement of the acetone content in the mixture of the solvents. In FIG. 2D, it should be noted that the mixing of the solvents A and B is carried out according to the same program curve shown in FIG. 2C. On the other hand, the problem of rounded shape of the actual mixing characteristic curve is not eliminated completely even in the case of FIG. 2D corresponding to the construction of the mixer 13 of FIG. 2B, indicating that there still exists a dead space 13x in the chamber 13b in which no substantial mixing of the solvents occurs.
FIGS. 3A and 3B show the construction of the sampler 14 used in the liquid chromatograph of FIG. 1.
Referring to FIG. 3A, the sampler 14 is essentially formed of a six-port valve including a rotary valve body 14R, wherein the rotary valve body 14R is formed with six passages .sup.14.sub.1 -14.sub.6, and there are formed three interconnecting passages 14a-14c each connecting two of the foregoing six passages 14.sub.1 -14.sub.6. In the illustrated state of FIG. 3A, the passage 14a connects the passages 14.sub.1 and 14.sub.6 with each other, the passage 14b connects the passages 14.sub.2 and 14.sub.3 with each other, and the passage 14c connects the passages 14.sub.5 and 14.sub.6 with each other. In the state of FIG. 3A, it should be noted that the passage 14.sub.1 is aligned to a port P that in turn is connected to the mixer 13, the passage 14.sub.2 is aligned to a part A.sub.1 connected to an end of a sample accumulation loop 14.sub.7 to be described later, the passage 14.sub.3 is aligned to a port S connected to the syringe 15, the passage 14.sub.4 is aligned to a port D connected to the waste reservoir 18, the passage 14.sub.5 is aligned to a port A.sub.2 connected to the other end of the loop 14.sub.7, and the passage 14.sub.6 is aligned to a port C connected to the column 16. As a result, the solvents A and B from the mixer 13 are supplied to the column 16 via the foregoing passages 14.sub.1, 14a and 14.sub.6, consecutively. In other words, only the solvents are supplied to the column in the state of FIG. 3A. No sample solution is supplied. Further, in the state of FIG. 3A, it should be noted that the syringe 15 communicates with the loop 14.sub.7 via the passages 14.sub.3, 14b and 14.sub.2 while the loop 14.sub.7 communicates with the waste reservoir 18 via the passages 14.sub.5, 14c and 14.sub.4. Thus, the sample solution is injected from the syringe 15 to the loop 14.sub.7 in the state of FIG. 3A and held therein.
In the state of FIG. 3B, on the other hand, the rotary valve body 14R is rotated in the direction of arrow, and as a result, the port P now aligns to the loop 14.sub.7 via the passages 14.sub.2, 14b and 14.sub.3. Further, the port C aligns to the passages 14.sub.1, 14a and 14.sub.6. As a result, the solvent supplied from the mixer 13 carries the sample solution, held in the loop 14.sub.7, to the column 16 via the port C. In other words, injection of the sample solution to the column 16 is carried out. Further, the syringe 15 is connected to the waste reservoir 18 via the passages 14.sub.4, 14c and 14.sub.5.
FIG. 4 shows the construction of the six-port valve 14 in an exploded view.
Referring to FIG. 4, the six-port valve 14 includes a stationary cap 14S acting as a stationary member on which the rotary valve body 14R is mounted rotatably. Further, the six-port valve 14 includes another valve body 14R.sub.2 that carries the foregoing passages 14a-14c. The rotary valve 14R carries thereon the passages 14.sub.1 -14.sub.6 in the form of straight, tubular passages, while the cap 14C carries the foregoing ports P, A.sub.1, S, D, A.sub.2 and C in alignment with the passages in the rotary valve body 14R. By rotating the rotary valve 14R with respect to the stationary cap 14S, the switching of the passage of fluid described with reference to FIGS. 2A and 2B is achieved.
In the six-port valve 14 of FIG. 4, it should be noted that the passages 14a-14c on the valve body 14R.sub.2 are generally formed with a tolerance such that passages 14.sub.1 -14.sub.6 align positively with the corresponding passages 14a-14c, even in the case where there exists an error in the precision of machining the passages. On the other hand, such a tolerance invites an increase in the size of the passages 14a-14c as indicated in FIG. 5, wherein it will be noted that a dead space 14ax is formed such that a part of the sample solution supplied to the loop 14.sub.7 from the mixer 13 dwells in such a dead space. Such a dead space 14ax inevitably invites incomplete supply of the sample solution to the column 16, and there occurs an error in the detection carried out by the detector 17.
In the liquid chromatograph of FIG. 1, it should further be noted that there exist a case in which collection of the sample solution separated by the column 16 is wished for various purposes. In order to meet such a demand, there is a construction of liquid chromatograph in which the sample solution is collected in a sample vessel after the analysis by the detector 17, rather than wasting the same to the waste reservoir 18, on the other hand, the sample solution obtained from the detector 17 is diluted by the solvents, and such a construction to recover the diluted waste solution generally requires an extensive facility. Thereby, the construction of the liquid chromatograph becomes inevitably large.
In the liquid chromatograph of FIG. 1, it should be noted that the rotary valve body 14R of FIG. 4 has been generally actuated by a stepping motor. Thus, the stepping motor rotates, in response to an external drive control signal, with a predetermined angle, and the switching of the fluid passage is achieved as a result. In the conventional six-port valve of FIG. 4, the cap 14S, the valve body 14R and the valve body 14R.sub.2 are all formed of metal in view of mechanical durability and durability against abrasion. Thus, the conventional liquid chromatograph requires an external controller such as a microcomputer for driving the stepping motor, and the construction for recovering the sample solution after the analysis can be realized only in those apparatuses having such a microcomputer as a controller. Further, because of the fact that the valve 14 is formed of metals, the conventional valve 14 is vulnerable to corrosion caused by the sample solution or solvents, and there has been a substantial risk that the result of analysis is unreliable due to the contamination caused by the corrosion of the valve 14. Further, the operation of the valve 14 may become unreliable due to the corrosion or wear.
FIG. 6 shows a sample injection needle 22 provided in the sampler 14 for injecting the sample solution to the column 16. In FIG. 6, the sample solution is indicated by a matting 30. Further, the injection needle 22 is used for injecting a cleaning solution 31 when the liquid chromatograph is in the cleaning mode. The needle 22 is provided at an end of a tube 32 extending to the syringe 15, wherein it will be noted that the connection between the needle 22 and the tube 32 is achieved by an intervening member 37. Conventionally, the cleaning solution is filled in the injection needle 22 as well as in the tube 32 prior to the injection of the sample solution 23, and a suction of the air is made to form an air gap 33 in the needle 22. Next, the suction of the sample solution 30 is made, and a suction of the air is made again to form a second air gap 34 in the needle 22. Thereby, it will be noted that the sample solution 30 is sandwiched by the air gaps 33 and 34 in the injection needle 22, and the injection of the sample solution 30 is made into the column 16 together with the gaps 33 and 34.
In the conventional process of sample injection shown in FIG. 6, it will be noted that the sample solution 30 may be adsorbed on the inner wall of the piping extending to the column 16, when an injection of the sample solution 30 is made. Thereby, there is a possibility that the sample solution 30 that reaches the column 16 and further to the detector 17 is lost by an amount corresponding to those adsorbed on the inner wall of the piping, and there is a problem that such a decrease of the sample causes an error in the result of the measurement. This problem of error becomes particularly pronounced in the chromatographs in which the amount of injected sample solution is small, in the order of 2 .mu.l or less.
FIG. 7 shows a conventional dual-column liquid chromatographic separation system, wherein there is provided a prefocusing column (precolumn) 40 between an automatic sampler 14 and a six-port switching valve 10, wherein the syringe 15 described before and a cooperating mechanism are collectively designated by the reference numeral 14. Further, a solvent-pumping system including the pumps 11A and 11B cooperates with the six-port valve 10. In the construction of FIG. 7, the precolumn 40 concentrates a sample or target substance that has been injected by the sample injector into a solvent A supplied by the pump 11A, and the substance thus concentrated is transferred further to the secondary column (separation column) 16 for separation together with a solvent B supplied by the pump 12. Thereby, the switching of the solvents A and B is achieved by the foregoing six-port valve 10. The transferred substances are separated and detected by the detector 17 cooperating with the separation column 16. Such a dual-column system is advantageous in view of elimination of sample treatment prior to the analysis and is commonly used as a "sample-treatment-free" system.
More specifically, the six-port valve 10 is switched between a first state and a second state, wherein, in the first state, the sample injected to the solvent A by the autosampler 14 is fed to the valve 10 after passing through the precolumn 40 and forwarded further to a waste reservoir along a path indicated by a continuous line, wherein the sample alone is captured by the precolumn 40. Simultaneously, the solvent B is supplied to the valve 10 and forwarded to the separation column 16 along another continuous line shown in FIG. 7.
In the second state, on the other hand, the concentrated sample from the precolumn 40 is now caused to flow to the separation column 16 along a path shown by a broken line in FIG. 7 for analysis, while the solvent B from the pump 2 is caused to flow to the waste reservoir along a path shown by another broken line. In such a conventional system, there has been a problem of dead volume in the valve 10. Because of the existence of such a dead volume, the switching of the fluid in the six-port valve 10 has not been complete.
It should be noted that the condensation column 40 is filled with a packing material that carries out the desired condensation of the sample solution. On the other hand, there is a tendency that such a packing material used in the condensation column 40 causes an adsorption of proteins, particularly when the liquid chromatograph is used for separation and analysis of mixtures that contain a large amount of proteins such as a serum. The adsorption of the proteins on the filler causes a substantial decrease in the effectiveness of the column filler for concentrating the sample solution. Conventionally, therefore, a process has been necessary for removing the proteins from the sample solution before it is supplied to the condensation column 40. Such a preparation of the sample solution, of course, is undesirable in view of extraneous time needed and possible degradation in the precision or reliability of the analysis.
Thus, there are proposals for the column filler for use in the condensation column 40 in which the necessity for removing proteins from the sample solution is eliminated. Generally, such improved column fillers are based upon porous glass or silica gel, and a material having a property different from that of the column filler is provided inside the minute pores of the filler. As a result, macro-molecules such as proteins in the serum cannot invade into the pores of the filler but simply pass through the condensation column 40 without being adsorbed upon the hydrophilic outer surface of the filler. Only small molecules such as the molecules of drugs are adsorbed on the hydrophobic inner surface of the pores.
The Japanese Laid-open Patent Publication 60-56256 describes an example of such an improved filler. In the filler disclosed in the foregoing prior art reference, a protein covers the outer surface of a silica body on which octadecylsilil (ODS) is bonded. The protein used herein may be bovine serum albumin and modifies the silica surface thus treated with ODS. In the conventional filler described above, however, there occurs a problem in that the adsorbed protein is generally released from the surface after prolonged use. Further, such a conventional filler cannot provide a column having high efficiency of separation.
In order to improve deficiency of such conventional column fillers, there are various proposals, as in the Japanese Laid-open Patent Publications 61-65159 and 1-123145, to treat the porous medium forming the filler by introducing hydrophobic groups upon the inner as well as outer surfaces of porous medium, and selectively disconnecting and removing the hydrophobic groups from the outer surface of the porous medium by means of enzyme. It should be noted that enzyme is a macro-molecule and cannot invade the interior of the minute pores of the medium forming the filler. Further, a hydrophilic group is introduced to the outer surface of the porous medium.
In the process of the foregoing '159 publication, in particular, a porous silica medium infiltrated with glyceryl propyl group is used as a starting material, and oligopeptide is bonded thereto via carbonyl diimidazole. Further, the phenylalanine side chains on the outer surface of the silica medium are disconnected by means of carboxypeptidase A, which is a proteoysis enzyme. As a result, the inner surface of the column filler is covered by glycyl-phenylalanyl phenyl-alanine acting as a hydrophobic ligand, while the outer surface of the filler is covered with hydrophilic glycyl-glyceryl propyl group.
In the process of the '145 reference, on the other hand, a porous silica body infiltrated with aminopropyl group is used as a starting material, and hydrophobic group is introduced by amide bonding by causing a reaction of octanoylchloride under presence of triethylamine. After this, acyl group on the outer surface of the silica filler is subjected to hydrolysis reaction, and amino group on the outer surface is made hydrophilic by causing a reaction with glycidol.
On the other hand, the column filler disclosed in the foregoing '159 or '145 reference has a drawback in that, because of use of enzyme reaction, the construction of the column filler becomes complex and the property of the obtained column filler tends to vary variously.
In the conventional liquid chromatograph of the foregoing references, there exists another drawback, associated with the large volumetric capacity of the condensation column 40, in that the sample solution supplied thereto may be unwantedly diluted in the column 40, rather than being concentrated. When this occurs, the sensitivity of detection decreases. It should be noted that the condensation column 40 generally has a volumetric capacity exceeding 200 .mu.l. This problem of deteriorated sensitivity of detection becomes particularly acute for those apparatuses designed for using small amount of sample solution.
Conventionally, it has been known that a better result is obtained when a micro-column having an inner diameter of less than 1 mm is used for the separation column 16, in place of a semi-micro-column that has an inner diameter of 1-2 mm. In fact, liquid chromatography analysis using a micro-column is described in various references such as Scott, R. P. and Kucera, P. J., J. Chromatogr., 125 (1978), pp.271, Tsuda, T. and Novotny, M., Anal. Chem. 50 (1978), pp.632, Ishii, D. et al., J. Chyromatogr., 124 (1977), pp.157, and Novotny, M., Anal. Chem. 60 (1988) 500A. These references generally stress the advantage of: (1) having a large theoretical number of stages, (2) improved response obtained, when a concentration-sensitive detector is used, due to the concentrating effect, (3) easiness for connecting the liquid chromatograph to other apparatuses that require removal of solvents such as mass spectrometer, and (4) reduced consumption of the solvents.
In spite of these various promising perspectives, practical use of the micro-column liquid chromatograph has not been successful so far, probably due to the instability in the flow rate control and the poor column stability. It should be noted that the stability of the isocratic is essential in the flow rate mode for managing the retention time. Further, such micro-column liquid chromatographs generally have a very basic problem of discrepancy between the volume of the prepared sample solution, typically in the order of several ten micro-litters to several hundred micro-litters, and the volume of the injected sample solution in the liquid chromatograph, which is typically in the order of several micro-litters. When the concentration level of the chemical substance to be analyzed is extremely low in the sample solution, it is naturally desired to use the entirely of the prepared sample solution for the analysis, while the conventional micro-column liquid chromatographs cannot meet such a demand.
In addition, it should be noted that the flow rate of the sample solution in the column 16 has to be set low, in the order of micro liters per minute when a micro-column is used. This level of flow rate is substantially smaller than those used in a semi-micro-column in which the flow rate is typically set to be about 0.05-0.2 ml/min. With reducing diameter of the column 16, the flow rate of the sample solution therein decreases further. Thus, in the construction of the liquid chromatograph of FIG. 7 where the condensation column 40 having a large diameter is connected directly to the micro-column 16, there inevitably occurs a problem of increased time for conducting the measurement. Thereby, the efficiency of analysis is substantially deteriorated.